Rickettsia species in Dermacentor reticulatus ticks feeding on human skin and clinical manifestations of tick-borne infections after tick bite

Dermacentor reticulatus ticks are sporadically removed from human skin and therefore the medical consequences of their feeding are neglected compared to Ixodes ricinus. We investigated the prevalence of pathogens in D. reticulatus removed from human skin and possible clinical manifestations suggestive of tick-borne diseases after a tick bite. A total of 2153 ticks were studied and of these only 34 were D. reticulatus. The mean prevalence of Rickettsia in D. reticulatus was 50.0% and R. raoultii was identified in 82.4% of infected D. reticulatus ticks. We confirmed the first case of R. aeschlimannii infection in D. reticulatus ticks. Among participants bitten by D. reticulatus, 13.3% reported reddening around the tick bite site and flu-like symptoms, including lymphadenopathy and 3.3% reported eschar on the tick site bite. All of the participants with flu-like symptoms after tick removal were bitten by ticks infected with R. raoultii. The results of this study indicate that even though D. reticulatus ticks bite humans sporadically, pathogenic Rickettsia have a remarkably high prevalence in this tick species. We can expect that the incidence of tick-borne lymphadenopathy might increase with the reported expansion of the D. reticulatus into new areas and its growing abundance in Central Europe.

role of this tick species in the life-cycle and transmission of these pathogenic microorganisms 13,16 . However, the low prevalence of some of these pathogens pose questions about the status of D. reticulatus as their vector.
The majority of published papers have focused on the pathogens prevalence in questing D. reticulatus ticks or engorged ticks collected from wildlife or domestic animals, mainly dogs. In rare situations, especially with a lack of other vertebrates in the area, people are chosen by Dermacentor reticulatus as a source of blood 17 . Dermacentor reticulatus ticks generally constitute less than 3% of all ticks removed from human skin 3,4,18 and therefore its role in pathogen transmission or medical consequences of a tick bite is often ignored. The studies about the D. reticulatus infestation in humans and the risk of clinical manifestation after tick bites, even if the rate of incidences is low, may be crucial among others for at-risk personnel, such as foresters, hunters, soldiers, and farmers. It is worth noting that it is a very expansive species, resistant to even hard environmental conditions, and year by year more often detected in cities 17,19 . As far as we are aware, no studies assessing the risk of possible symptoms of tick-borne infection in humans after D. reticulatus feeding have previously been carried out in Europe. The main aims of the present study were to investigate the prevalence of tick-borne pathogens in D. reticulatus ticks removed from human skin and possible symptoms and clinical manifestations suggestive of tick-borne diseases after a tick bite.

Results
Dermacentor reticulatus ticks. A total of 2153 ticks removed by participants during 2021-2022 could be further investigated, and of these only 34 (1.6%) were D. reticulatus. Of the 34 feeding D. reticulatus, 2 (5.9%) were nymphs, 25 (73.5%) were females, and 7 (20.6%) were males. The molecular studies have confirmed the morphological identification of two D. reticulatus nymphs. The nucleotide sequences of the cox1 gene fragment of tested nymphs were identical and highly similar (99.9%; 778/779) to the sequence originally obtained from D. reticulatus in Czech Republic (OM142141; 20 ) and Novosibirsk region in Russia (OM867328). Most D. reticulatus ticks were collected from September to November (21; 61.8%). Only 6 (18.2%) ticks were removed in July and August, however, the majority of them (5 out of 6) were collected between the 20th and 31st of August. The rest of the ticks (7), including two nymphs, were brought to the laboratory in May and in the first half of June.
Only one D. reticulatus tick was removed from the skin of each participant (n = 34). The data about among others sex and age of participants, anatomical location of tick bites, and flu-like symptoms after tick bite were collected and then analysed on the basis of filled-out questionnaires at enrolment ( (Fig. 2). Single D. reticulatus ticks were also found on the stomach, back and groin.
Description of the Dermacentor reticulatus bitten participants. More women than men participated in this study (22/33; 66.7% vs. 11/33; 33.3%) and the mean age of participants was 34 years (range . The majority of the study individuals (90.9%; 30/33) have declared that they did not use chemoprophylaxis (repellents) against ticks. However, 78.8% (26/33) of all participants have admitted wearing protective suits (i.e. long sleeves and legs, headgear, tucking the pants into long socks) when they were bitten by ticks. Three out of 33 participants (9.1%) were blood donors.

Self-reported non-specific symptoms among the participants after 8 weeks of tick bite.
In total 30 study participants removed D. reticulatus ticks of which 1 (3.3%) reported reddening around the tick bite site, 2 (6.7%) reported flu-like symptoms and 4 (13.3%) reported both of them. One participant (3.3%) reported eschar ('tache noir') diagnosed by a general practitioner (GP) on the R. raoultii-infected tick bite site. Reddening at the bite site did not show the pattern of erythema migrans in any of the cases on the basis of GP diagnosis. Flu-like symptoms included fever (n = 6), headache (n = 6); malaise (n = 3), fatigue (n = 3), muscle pain (n = 2) and lymphadenopathy on the neck (n = 2). Interestingly, all of the participants who reported flu-like symptoms after tick removal were bitten by D. reticulatus ticks infected with R. raoultii (χ 2 1 = 8.85; p = 0.003). No significant differences were observed in the frequency of reddening at the bite site presence among participants bitten by Rickettsia-infected and non-infected ticks (p = 0.176).

Discussion
To our knowledge, this is the first study on the risk of possible symptoms suggestive of tick-borne infection in humans after D. reticulatus removal from the skin. The main limitation of our study was the low number of collected ticks, however, adults D. reticulatus feed mainly on wild ungulates whereas immature forms-on rodents, and therefore they are sporadically removed from human skin 24 . Nevertheless, during the last two decades, an increasing number of studies has reported significant habitat expansion of D. reticulatus ticks in several European countries, including Poland 10,17,25,27 . The geographical range expansion of D. reticulatus ticks is concerning in light of their vector potential and high prevalence of tick-borne pathogens i.e. Rickettsia.
In our present and previous studies 3 , D. reticulatus ticks showed a bimodal activity pattern, with the highest density in May-June (the first half) and September-November, whereas only single ticks were collected in the summer (end of August), which is typical for this tick species 28,29 . Since insectivores and small rodents (mostly voles) are mainly hosts for larvae and nymphs and for adult ticks-medium-sized mammals (carnivores, sheep, goats, deer, cattle, and European bison) 24 , the risk of being bitten by D. reticulatus is significantly higher on suburban and rural areas. However, this pattern is likely to be characteristic also for I. ricinus tick species 30 . In our study women removed a greater proportion of D. reticulatus ticks compared to men which might reflect morphological, behavioral, and physiological differences between men and women 31 . Wilhelmsson and co-authors 31 have suggested that men and women can differ in their capacity to rapidly detect the tick on the skin due to more hairy skin of the men and/or their grooming behavior. Moreover, it has been shown that women have a higher  www.nature.com/scientificreports/ risk perception concerning tick bites that could influence the protective behaviour 32,33 . Nonetheless, in our study the majority of the participants (> 90%) have declared not to use chemoprophylaxis against ticks. It could be the result of disliking the idea of applying a chemical to bodies (especially in case of children), perceiving repellents as unsafe, and the frequency (usually every 4-6 h) or method of application of tick repellents 33,34 . Simultaneously, almost 80% of respondents have admitted wearing protective suits when they were bitten by ticks, which seems to be an insufficient protection measure. It is believed that humans are likely to be bitten by different tick species in potentially various anatomical sites and in varying seasonality 2,35 . In our study, results should be interpreted with caution due to significant discrepancies in the number of groups of both tick species, however some differences in ticks' most likely location could be observed. All D. reticulatus life stages were found significantly more frequently on the head (scalp) compared to I. ricinus ticks. Despite containing less than 10% of the surface area of the human body, the head was identified as the attachment point of almost 50% of all D. reticulatus ticks. The authors of previous studies 2, 36 have suggested that especially children are prone to be bitten by ticks on the head and/or neck due to behavioral (frequent enter vegetation while playing) and physiological (head/neck on the level of vegetation with questing ticks) differences between adults and children. In our study only 5 D. reticulatus ticks (15.2%; 5/33) were removed from children under 15 years, however the majority of them (4/5; 80.0%) were collected from the head (scalp). Therefore, it seems that adult D. reticulatus ticks, parasitizing mainly medium-sized mammals, prefer the hairy skin of the head. It also corresponds with the fact that the majority (> 93%) of patients affected by Dermacentor-Borne-Necrosis-Erythema-Lymphadenopathy (DEBONEL) were bitten on the scalp 37 . According to Hart and co-workers 38 , this is also clear evidence of the climbing behaviour of Dermacentor ticks. It is likely that hair protects them from being immediately detected and removed by animal hosts, obscuring them until they can feed extensively 38 . It is also worth noting that the tick site attachment to the human body might be of clinical importance 31 . Significantly greater proportion of neurological manifestation of Lyme borreliosis (LB) among patients who had been bitten on the head or neck was shown than among LB patients bitten on other parts of their body 39 . Therefore, understanding ticks' most likely location can be useful for removing them to minimise pathogens transmission or diagnostically to confirm the presence of the tick.
Despite the fact that D. reticulatus ticks constituted less than 3% of all ticks collected from human skin (1.6% in our study) 2,40 they play the key role as the Rickettsia vectors. In our study prevalence of Rickettsia in D. reticulatus ticks was 50% whereas no A. phagocytophilum, B. burgdorferi s.l., Babesia spp. and N. mikurensis DNA were detected. Our results of Rickettsia prevalence correspond with our previous study 16 as well as with other data from Poland 41,42 , Germany 43 , Hungary 44 and Slovakia 11 . Not surprisingly, the majority of all Rickettsia isolates (14 out of 17) were identified as R. raoultii. Rickettsia helvetica is identified rarely in D. reticulatus compared to R. raoultii 45,46 and I. ricinus ticks serve as the main vector for this pathogen species. According to our best knowledge, we have confirmed for the first time the presence of R. aeschlimannii in D. reticulatus ticks. Rickettsia aeschlimannii was described in 1997 as the new spotted fever group Rickettsia associated with Hyalomma marginatum ticks 47 . Rickettsia aeschlimannii is an emerging human and animal pathogen, reported from various ticks in Europe and Africa, including several Hyalomma spp. ticks collected from migrant bird species 33,48 , which might explain the presence of this pathogen in ticks collected in Poland. The first human infection caused by R. aeschlimannii was reported for a French patient who became ill after returning from Morocco and who exhibited symptoms similar to those of Mediterranean Spotted Fever 48 . First human cases of R. aeschlimannii infection were recently noted in Russia and China 49,50 . In our study, no symptoms of tick-borne infection were declared by a participant who was bitten by R. aeschlimannii-infected tick. However, the role of D. reticulatus as a vector of R. aeschlimannii in Europe need further, urgent investigation.
Several cases of DEBONEL/TIBOLA have been described so far in Europe, including Poland 14,15,51-54 . At the site of Dermacentor tick bite, a high percentage of patients develop an inoculation eschar (necrosis) surrounded by an erythema and regional enlarged and painful lymphadenopathies 55 . In 40% of patients affected by DEBONEL fever was also observed 37 . Here, the flu-like symptoms (fever, headache, malaise, fatigue and muscle pain) were noted in 6 out of 30 study participants (20%), including two respondents who simultaneously reported lymphadenopathy on the neck. All participants who declared flu-like symptoms (including lymphadenopathy on the neck) after tick removal were bitten by D. reticulatus ticks infected with R. raoultii what might suggest the Rickettsia infection. However, further serological studies are needed. In our study, 2 out of 3 blood donors were bitten by R. raoultii infected ticks and one of them reported flu-like symptoms. Several tick-borne pathogens can potentially be transmitted through blood transfusion, i.e. Anaplasma phagocytophilum or Babesia microti 56 . Many tick-borne microorganisms are located intracellularly, which is an excellent condition for transmission by transfusion 56 . The risk of transfusion-transmitted Rickettsia is unknown, however, single cases of R. rickettsia and R. parkeri have been described so far 57,58 . Lucchese and co-authors 59 have demonstrated that R. conorii was able to remain viable in dog blood stored until 35 days. Therefore further studies about the risk of transfusiontransmitted Rickettsia infection are essential, especially if we take into account that blood recipients are often immunosuppressed 60 .
In our study in the case of one participant, Rickettsia infection was diagnosed by GP on the basis of the black eschar on the tick bite site and then treated with amoxicillin 500 mg three times a day for 7 days (data unpublished). The eschar surrounded by erythema appeared 24 h after tick removal and the improvement of skin condition was observed after 48 h of antibiotic treatment. The tick was identified as D. reticulatus females infected with R. raoultii. According to the recommendations of The Polish Society of Epidemiology and Infectious Diseases, the laboratory criteria for rickettsiosis confirmation include the detection of a fourfold increase in antibody titre in sera during the acute and recovery phase of infection or detection of Rickettsia DNA in blood/ eschar 61 . In our study, serological and/or molecular tests were not ordered by GP, therefore confirmation of rickettsiosis according to recommendations was not possible.

Conclusion
This study describes the ticks infestation pattern, Rickettsia prevalence and possible medical consequences of D. reticulatus feeding on human skin. Information about tick infestation patterns provided by this study should be valuable for predicting the biting location of ticks dependent on species and potentially reducing the transmission of tick-borne pathogens by quick tick removal. The results of this study indicate that even though D. reticulatus bite human sporadically, pathogenic rickettsial species have a remarkably high prevalence in this tick species. We have also confirmed the first case of R. aeschlimannii infection in D. reticulatus female. Noteworthy is that all participants reporting non-specific, flu-like symptoms, including lymphadenopathy, after tick removal were bitten by D. reticulatus infected by R. raoultii, and in one case rickettsiosis was confirmed by GP based on clinical manifestation (eschar). We can expect that the incidence of tick-borne lymphadenopathy (DEBONEL/ TIBOLA) might increase with the reported expansion of the D. reticulatus vector into new areas and its growing abundance in Central Europe.

Materials and methods
Ethics approval and consent to participate. The Internal Review Board of the Warsaw Medical University was informed about the study protocol (no. AKBE/73/2021). The study protocol followed ethical guidelines of the 2013 Declaration of Helsinki. Informed consent was obtained from all individual participants included in the study.
Study design. The research reported here was conducted over a 2-year period in 2021 and 2022. The information about our study was disseminated on the University of Warsaw website and on the websites dedicated to medicine, diagnostics, and health care, in social networks, as well as among the university society through email (researchers, students, and administration workers). The study subjects were asked to bring or send their tick(s) to the Department of Parasitology, University of Warsaw in a tightly-sealed, ethanol-filled container within 5 days after removal of the tick(s) from the skin by a physician or the patients themselves. Each patient was included in the study after an informed consent and received information on the aims and the protocol of the study. In case of patients under the legal age of consent (< 18 years old), one of the parents signed the agreement.
Questionnaires. The study participants were asked to fill out an online questionnaire at enrolment which included questions on the number of tick bite(s), where the tick(s) was/were encountered (urban/rural), the use of chemoprophylaxis and/or protective suits. All the patients were also asked for follow-up after 8 weeks by mail or telephone. The second questionnaire consisted the questions about new tick bites and the person's general health condition during the past two months, reddening at the bite site, symptoms possibly associated with tick-borne diseases, including erythema migrans, medical records from participants who attended general practitioner/infectious diseases specialist appointments for symptoms possibly associated with tick-borne diseases, questions about the results of serological test for Lyme borreliosis (if such were performed) as well as antibiotic treatment within the last 8 weeks. The individuals who reported a second bite within the 8 weeks from first notification and the patients with immunosuppression were excluded from further study.
Tick collection and identification. The ticks were collected throughout Poland from March to November of each year (2021, 2022). Ticks were morphologically identified in terms of species and developmental stage using a standard taxonomic key 62 . Specimens that could not be identified due to being extensively damaged when being removed from the skin were not included in the study. To confirm morphological identification of D. reticulatus nymphs, the fragment of the cytochrome c oxidase subunit I (cox1) was amplified and sequenced 63  www.nature.com/scientificreports/ using maximum likelihood techniques based on log-linear analysis of contingency tables (HILOGLINEAR). For analysis of the prevalence of Rickettsia in ticks, we fitted the prevalence of pathogens as a binary factor (infected = 1, uninfected = 0) and then by year (2 levels: 2021-2022), tick stadium (female and male) and type of area (urban and rural). For analysis of the associations between Rickettsia DNA detected in ticks or tick species and the patient-reported data at enrolment and after two months, we used the same statistical approach. We fitted the Rickettsia prevalence as a binary factor (infected = 1, uninfected = 0) and then by among others selfreported flu-like symptoms (fatigue, headache, loss of appetite, loss of weight, nausea, fever, neck pain, loss of appetite, vertigo, cognitive difficulties, radiating pain, mylagia/ arthralgia, numbness), cutaneous manifestations (local reaction to tick bite) and erythema migrans or eschar ('tache noir') diagnosed by a general practitioner (GP) or infectious diseases specialist (present and not present). We also fitted the tick species as a binary factor (I. ricinus = 1, D. reticulatus = 2) and then by sex, immunosuppression, site of tick attachment, number of ticks detached, where the tick was encountered, estimated time of tick attachment, previous tick bites, and use of chemoprophylaxis/ protective suits. P values < 0.05 were considered to be statistically significant.
Ethics declarations. The Internal Review Board of the Warsaw Medical University was informed about the study protocol (no. AKBE/73/2021). The study protocol followed ethical guidelines of the 2013 Declaration of Helsinki. Informed consent was obtained from all individual participants included in the study.

Data availability
The datasets used and analysed during this study are available from the corresponding author (RWF) on reasonable request. The new nucleotide sequences of Rickettsia species have been deposited in the GenBank database under accession numbers OQ689706-OQ689708 and OR000446-OR000448.